Two Gram-negative, nonmotile, coccobacilli, SW- and SW-, were isolated from sea water of the Yellow Sea in Korea. Strains SW- and SW- contained ubiquinone-9 (Q-9) as the predominant respiratory lipoquinone and and as the major fatty acids. The DNA G+C contents of strains SW- and SW- were 44.1 mol% and 41.9 mol%, respectively. A neighbor-joining tree based on l6S rRNA gene sequences showed that the two isolates fell within the evolutionary radiation enclosed by the genus Acinetobacter. Strains SW- and SW- exhibited a l6S rRNA gene similarity value of 95.7% and a mean DNA-DNA relatedness level of 9.2%. Strain SW- exhibited l6S rRNA gene sequence similarity levels of 93.5-96.9% to the validly described Acinetobacter species and fifteen Acinetobacter genomic species. Strain SW- exhibited l6S rRNA gene sequence similarity levels of less than 97.0% to the other Acinetobacter species except Acinetobacter towneri DSM (98.0% similarity). Strains SW- and SW- exhibited mean levels of DNA-DNA relatedness of 7.3-l6.7% to the type strains of some phylogenetically related Acinetobacter species. On the basis of phenotypic, phylogenetic, and genetic data, strains SW- and SW- were classified in the genus Acinetobacter as two distinct novel species, for which the names Acinetobacter marinus sp. novo (type strain SW-=KCTC =DSM ) and Acinetobacter seohaensis sp. novo (type strain SW-=KCTC =DSM ) are proposed, respectively.

Elastin-like polypeptides (ELPs) undergo a reversible inverse phase transition upon a change in temperature. This thermally triggered phase transition allows for a simple and rapid means of purifying a fusion protein. Recovery of ELPs-tagged fusion protein was easily achieved by aggregation, triggered either by raising temperature or by adding salt. In this study, levansucrase has been used as a model enzyme in the development of a simple one-step purification method using ELPs. The levansucrase gene cloned from Pseudomonas aurantiaca S-4380 was tagged with various sizes of ELPs to functionally express and optimize the purification of levansucrase. One of two ELPs, ELP[V-20] or ELP[V-40], was fused at the C-terminus of the levansucrase gene. A levansucrase-ELP fusion protein was expressed in Escherichia coli at for 18 h. The molecular masses of levansucrase-ELP[V-20] and levansucrase-ELP[V-40] were determined as 56 kDa and 65 kDa, respectively. The phase transition of levansucrase-ELP[V-20] occurred at in 50 mM Tris-Cl (pH 8) buffer with 3 M NaCl added, whereas the phase transition temperature () of levansucrase-ELP[V-40] was with 2 M NaCl. Levansucrase was successfully purified using the phase transition characteristics of ELPs, with a recovery yield of higher than 80%, as verified by SDS-PAGE. The specific activity was measured spectrophotometrically to be 173 U/mg and 171 U/mg for levansucrase-ELP[V-20] and levansucrase-ELP[V-40], respectively, implying that the ELP-tagging system provides an efficient one-step separation method for protein purification.

Synthesis of oligosaccharides during milk fermentation was attempted by inoculating Leuconostoc citreum with Lactobacillus casei, Lb. delbrueckii subsp. bulgaricus, and Streptococcus thermophilus as starters. Dextransucrase of Ln. citreum worked as a catalyst for the transglycosylation reaction of sugars; sucrose was added as the glucose donor, and lactose or maltose acted as the acceptor compound for the reaction. When 4% sucrose was added in milk, glucosyl-lactose was synthesized (about 1%, w/v) after 1-2 days of fermentation at 15 or . Alternatively, when sucrose and maltose (2% each, w/v) were added, panose (about 1 %, w/v) and other isomaltooligosaccharides were made in a day at . Growth patterns of lactobacilli and streptococci starters were not affected by the coculture of leuconostoc starter, but the rate of acid synthesis was slightly slowed at every temperature. Addition of sugars in milk did not give any adverse effect on the lactate fermentation. Accordingly, the use of leuconostoc starter and addition of sugars in milk allowed the production of oligosaccharides-containing fermented milk, and application of this method will facilitate the extensive development of synbiotic lactate foods.

A polymerase chain reaction (PCR)-based method was developed to detect the DNA of Ralstonia solanacearum, the causal agent of bacterial wilt in various crop plants. One pair of primers (RALSF and RALSR), designed using cytochrome c1 signal peptide sequences specific to R. solanacearum, produced a PCR product of 932 bp from 13 isolates of R. solanacearum from several countries. The primer specificity was then tested using DNA from 21 isolates of Ralstonia, Pseudomonas, Burkholderia, Xanthomonas, and Fusarium oxysporum f. sp. dianthi. The specificity of the cytochrome c1 signal peptide sequences in R. solanacearum was further confirmed by a DNA-dot blot analysis. Moreover, the primer pair was able to detect the pathogen in artificially inoculated soil and tomato plants. Therefore, the present results indicate that the primer pair can be effectively used for the detection of R. solanacearum in soil and host plants.

Activated sludge was sequentially adapted to benzene, toluene, and o-xylene (BTX) to study the effects on the change of microbial community. Sludge adapted to BTX separately degraded each by various rates in the following order; toluene>o-xylene>benzene. Degradation rates were increased after exposure to repeated spikes of substrates. Eleven different kinds of sludge were prepared by the combination of BTX sequential adaptations. Clustering analyses (Jaccard, Dice, Pearson, and cosine product coefficient and dimensional analysis of MDS and PCA for DGGE patterns) revealed that acclimated sludge had different features from nonacclimated sludge and could be grouped together according to their prior treatment. Benzene- and xylene-adapted sludge communities showed similar profiles. The sludge profile was affected from the point of the final adaptation substrate regardless of the adaptation sequence followed. In the sludge adapted to 50 ppm toluene, Nitrosomonas sp. and bacterium were dominant, but these bands were not dominant in benzene and benzene after toluene adaptations. Instead, Flexibacter sp. was dominant in these cultures. Dechloromonas sp. was dominant in the culture adapted to 50 ppm benzene. Thauera sp. was the main band in the sludge adapted to 50 ppm xylene, but became vaguer as the xylene concentration was increased. Rather, Flexibacter sp. dominated in the sludge adapted to 100 ppm xylene, although not in the culture adapted to 250 ppm xylene. Two bacterial species dominated in the sludge adapted to 250 ppm xylene, and they also existed in the sludge adapted to 250 ppm xylene after toluene and benzene.

Cellulosomes in Clostridium cellulovorans are assembled by the interaction between the repeated cohesin domains of a scaffolding protein (CbpA) and the dockerin domain of enzyme components. In this study, we determined the synergistic effects on cellulosic and hemicellulosic substrates by three different recombinant mini-cellulosomes containing either endoglucanase EngB or endoxylanase XynA bound to mini-CbpA with one cohesin domain (mini-CbpAl), two cohesins (mini-CbpA12), or four cohesins (mini-CbpAl234). The assembly of EngB or XynA with mini-CbpA increased the activity against carboxymethyl cellulose, acid-swollen cellulose, Avicel, xylan, and com fiber 1.1-1.8-fold compared with that for the corresponding enzyme alone. A most distinct improvement was shown with com fiber, a natural substrate containing xylan, arabinan, and cellulose. However, there was little difference in activity between the three different mini-cellulosomes when the cellulosomal enzyme concentration was held constant regardless of the copy number of cohesins in the cellulosome. A synergistic effect was observed when the enzyme concentration was increased to be proportional to the number of cohesins in the mini-cellulosome. The highest degree of synergy was observed with mini-CbpAl234 (1.8-fold) and then mini-CbpAl2 (1.3-fold), and the lowest synergy was observed with mini-CbpAl (1.2-fold) when Avicel was used as the substrate. As the copy number of cohesin was increased, there was more synergy. These results indicate that the clustering effect (physical enzyme proximity) of the enzyme within the mini-cellulosome is one of the important factors for efficient degradation of plant cell walls.

A -glucosidase with the molecular mass of 160,000 Da was purified to homogeneity from cell extract of a cellulolytic bacterium, Cellulomonas uda CS1-1. The kinetic parameters ( and ) of the enzyme were determined with pNP-cellooligosccharides (DP 1-5) and cellobiose. The molecular orbital theoretical studies on the cellulolytic reactivity between the pNP-cellooligosaccharides as substrate (S) molecules and the purified -glucosidase (E) were conducted by applying the frontier molecular orbital (FMO) interaction theory. The results of the FMO interaction between E and S molecules verified that the first stage of the reaction was induced by exocyclic cleavage, which occurred in an electrophilic reaction based on a strong charge-controlled reaction between the highest occupied molecular orbital (HOMO) energy of the S molecule and the lowest occupied molecular orbital (LUMO) energy of the hydronium ion (), more than endocyclic cleavage, whereas a nucleophilic substitution reaction was induced by an orbital-controlled reaction between the LUMO energy of the oxonium ion () protonated to the S molecule and the HOMO energy of the molecule. A hypothetic reaction route was proposed with the experimental results in which the enzymatic acid-catalyst hydrolysis reaction of E and S molecules would be progressed via and reactions. In addition, the quantitative structure-activity relationships (QSARs) between these kinetic parameters showed that has a significant correlation with hydrophobicity (logP), and specific activity has with dipole moment, respectively.

Lycopene, an acyclic carotenoid found in tomatoes (Lycopersicon esculentum) and a number off fruits, has shown various biological properties, but its antifungal effects remain poorly understood. The current study investigated the antifungal activity of lycopene and its mode of action. Lycopene showed potent antifungal effects toward pathogenic fungi, tested in an energy-independent manner, with low hemolytic effects against human erythrocytes. To confirm the antifungal effects of lycopene, its effects on the dimorphism of Candida albicans induced by fetal bovine serum (FBS), which plays a key role in the pathogenesis of a host invasion, were investigated. The results showed that lycopene exerted potent antifungal activity on the serum-induced mycelia of C. albicans. To understand the antifungal mode of action of lycopene, the action of lycopene against fungal cell membranes was examined by FACScan analysis and glucose and trehalose-release test. The results indicated that lycopene caused significant membrane damage and inhibited the normal budding process, resulting from the destruction of membrane integrity. The present study indicates that lycopene has considerable antifungal activity, deserving further investigation for clinical applications.

Purple nonsulfur bacteria were isolated from river sediments and their growth promoting capabilities on tomato were examined. Isolated strains KL9 and BL6 were identified as Rhodopseudomonas spp. by 16S rDNA sequence analysis. Rhodopseudomonas strain KL9 maximally produced 5.56 mM/min/mg protein and protein of indole-3-acetic acid (IAA) and 5-aminolevulinic acid (ALA), respectively, which may be one of the mechanisms of plant growth enhancement. The germination percentage of tomato seed, total length, and dry mass of germinated tomato seedling increased by 30.2%, 71.1%, and 270.8%, respectively, compared with those of the uninoculated control 7 days after inoculation of strain KL9. The lengths of the root and shoot of germinated seedling treated with 3 mM tryptophan, a precursor of IAA, increased by 104.4% and 156.5%, respectively, 7 days after inoculation of strain KL9. Rhodopseudomonas KL9 increased 123.5% and 54% of the root and shoot lengths of germinated seedling, respectively, treated with 15 mM glycine and succinate, precursors of ALA. This plant growth promoting capability of purple nonsulfur bacteria may be a candidate for a biofertilizer in agriculture.

Extracellular enzymes from Lentinus edodes M290 on normal woods (Quercus mongolica) and waste logs from oak mushroom production were comparatively investigated. Endoglucanase, cellobiohydrolase, -glucosidase, and xylanase activities were higher on waste mushroom logs than on normal woods after 1. edodes M290 inoculation. Xylanase activity was especially different, with a three times higher activity on waste mushroom logs. When the waste mushroom logs were used as a carbon source, a new 35 kDa protein appeared. After the purification, the optimal pH and temperature for xylanase activity were determined to be 4.0 and , respectively. More than 50% of the optimal xylanase activity was retained when the temperature was increased from 20 to , after a 240 min reaction. At , the xylanase maintained 93% of the optimal activity, after a 240 min reaction. The purified xylanase showed a very high homology to the xylanase family 10 from Aspergillus terreus by LC/MS-MS analysis. The highest Xcorr (1.737) was obtained from the peptide KWI SQGIPIDGIG SQTHLGSGGS WTVK originated from Aspergillus terreus, indicating that the 35 kDa protein was xylanase. This protein showed low homology to a previously reported L. edodes xylanase sequence.

S-Adenosylmethionine (SAM) was previously documented to activate secondary metabolism in a variety of Streptomyces spp. and to promote actinorhodin (ACT) and undecylprodigiosin (RED) in Streptomyces coelicolor. The SAM-induced proteins in S. coelicolor include several ABC transporter components (SCO5260 and SCO5477) including BldKB, the component of a well-known regulatory factor for differentiations. In order to assess the role of these ABC transporter complexes in differentiation of Streptomyces, SCO5260 and SCO5476, the first genes from the cognate complex clusters, were individually inactivated by gene replacement. Inactivation of either SCO5260 or SCO5476 led to impaired sporulation on agar medium, with the more drastic defect in the SCO5260 null mutant (). displayed growth retardation and reduced yields of ACT and RED in liquid cultures. In addition, SAM supplementation failed in promoting the production of ACT and RED in . Inactivation of SCO5476 gave no significant change in growth and production of ACT and RED, but impaired the promoting effect of SAM on ACT production without interfering with the effect on RED production. The present study suggests that SAM induces several ABC transporters to modulate secondary metabolism and morphological development in S. coelicolor.

We describe the fabrication of poly(ethylene glycol) diacrylate (PEG-DA) hydrogel microstructures with a high aspect ratio and the use of hydrogel microstructures containing the enzyme -galactosidase (-Gal) or glucose oxidase (GOx)/horseradish peroxidase (HRP) as biosensing components for the simultaneous detection of multiple analytes. The diameters of the hydrogel microstructures were almost the same at the top and at the bottom, indicating that no differential curing occurred through the thickness of the hydrogel microstructure. Using the hydrogel microstructures as microreactors, -Gal or GOx/HRP was trapped in the hydrogel array, and the time-dependent fluorescence intensities of the hydrogel array were investigated to determine the dynamic uptake of substrates into the PEG-DA hydrogel. The time required to reach steady-state fluorescence by glucose diffusing into the hydrogel and its enzymatic reactions with GOx and HRP was half the time required for resorufin -D-galactopyranoside (RGB) when used as the substrate for -Gal. Spatially addressed hydrogel microarrays containing different enzymes were micropatterned for the simultaneous detection of multiple analytes, and glucose and RGB solutions were incubated as substrates. These results indicate that there was no cross-talk between the -Gal-immobilizing hydrogel micropatches and the GOx/HRP-immobilizing micropatches.

Viruses in garlic plants (Allium sativum L.) have accumulated and evolved over generations, resulting in serious consequences for the garlic trade around the world. These viral epidemics are also known to be caused by aphids and eriophyid mites (Aceria tulipae) carrying Potyviruses, Carlaviruses, and Allexiviruses. However, little is known about viral epidemics in garlic plants caused by eriophyid mites. Therefore, this study investigated the infection of garlic plants with Allexiviruses by eriophyid mites. When healthy garlic plants were cocultured with eriophyid mites, the leaves of the garlic plants developed yellow mosaic strips and became distorted. In extracts from the eriophyid mites, Allexiviruses were observed using immunosorbent electron microscopy (ISEM). From an immunoblot analysis, coat proteins against an Allexivirus garlic-virus antiserum were clearly identified in purified extracts from collected viral-infected garlic plants, eriophyid mites, and garlic plants infected by eriophyid mites. A new strain of GarV-B was isolated and named GarV-B Korea isolate 1 (GarV-B1). The ORF1 and ORF2 in GarV-B1 contained a typical viral helicase, RNA-directed RNA polymerase (RdRp), and triple gene block protein (TGBp) for viral movement between cells. The newly identified GarV-B1 was phylogenetically grouped with GarV-C and GarV-X in the Allexivirus genus. All the results in this study demonstrated that eriophyid mites are a transmitter insect species for Allexiviruses.

We previously identified the origin of replication of p703/5, a small cryptic plasmid from the KBL703 strain of Enterococcus faecalis. The origin of replication contains putative regulatory cis-elements required for replication and a replication initiator (RepA) gene. The replicon of p703/5 is similar in its structural organization to theta-type plasmids, and RepA is homologous to a family of Rep proteins identified in several plasmids from Gram-positive bacteria. Here, we report molecular interactions between RepA and the replication origin of p703/5. DNase I footprinting using recombinant RepA together with electrophoretic mobility shift assays confirmed the binding of RepA to the replication origin of p703/5 via iterons and an inverted repeat. We also demonstrated the formation of RepA dimers and the different binding of RepA to the iteron and the inverted repeat using gel filtration chromatographic analysis, a chemical crosslinking assay, and electrophoretic mobility shift assays in the presence of guanidine hydrochloride. Our results suggest that RepA plays a regulatory role in the replication of the enterococcal plasmid p703/5 via mechanisms similar to those of typical iteroncarrying theta-type plasmids.

Antibacterial activity of essential oils (Tea tree, Chamomile, Eucalyptus) on Staphylococcus aureus growth was evaluated as well as the essential oil-loaded alginate beads. The binding interactions between the cell and the essential oils were measured using an optical biosensor. The antibacterial activity of the essential oils to the cell was evaluated with their binding interaction and affinity. The antibacterial activity appeared in the order of Tea Tree>Chamomile>Eucalyptus, in comparison of the inhibition effects of the cell growth to the essential oils. The association rate constant and affinity of the cell binding on Tea Tree essential oil were and , respectively. The affinity of the cell binding on Tea Tree was about twice higher than those on the other essential oils. It might be possible that an effective antibacterial activity of Tea Tree essential oil was derived from its strong adhesive ability to the cell, more so than those of the other essential oils.

Physiological cell conditions such as glucose deprivation and hypoxia play roles in the development of drug resistance in solid tumors. These tumor-specific conditions cause decreased expression of DNA topoisomerase , rendering cells resistant to topo II target drugs such as etoposide. Thus, targeting tumor-specific conditions such as a low glucose environment may be a novel strategy in the development of anticancer drugs. On this basis, we established a novel screening program for anticancer agents with preferential cytotoxic activity in cancer cells under glucose-deprived conditions. We recently isolated an active compound, AA-98, from Streptomyces sp. AA030098 that can prevent stress-induced etoposide resistance in vitro. Furthermore, LC-MS and various NMR spectroscopic methods identified AA-98 as mithramycin, which belongs to the aureolic acid group of antitumor compounds. We found that mithramycin prevents the etoposide resistance that is induced by glucose deprivation. The etoposide-chemosensitive action of mithramycin was just dependent on strict low glucose conditions, and resulted in the selective cell death of etoposide-resistant HT-29 human colon cancer cells.

An individual's immune response is critical for host protection from many different pathogens, and the responsiveness can be assessed by the amount of cytokine production upon stimulating bacterial components such as lipopolysaccharide (LPS). The difference between individuals in their peripheral blood mononuclear cells (PBMC) responsiveness to LPS, a Gram-negative endotoxin, was investigated from 27 healthy individuals. We observed a large variation in production among different individuals. The PBMC of the consistently three highest and three lowest producers were investigated. Since previous studies described that a single point mutation in the coding region of TLR2 and TLR4 is linked to the individual responsiveness to pathogenic bacterial infections, we first examined the known point mutations in the coding region of , located in the cytoplasmic regions of the Toll-like domain as well as located in the extracellular region. None of these mutations were associated with an individual's responsiveness to LPS, despite the presence of mutation. Further investigation revealed that the variation of PBMC responsiveness to LPS among healthy individuals was due to constitutive expression levels of TLR4 and TLR2. This result is consistent with an aging-related low expression of Toll-like receptors in the mouse model of LPS responsiveness. The present study therefore suggests that the constitutive expression levels of TLR2 and TLR4 may contribute to the individual response to LPS.

We have developed a robotic system for an automated parallel cell cultivation process that enables screening of induction parameters for the soluble expression of recombinant protein. The system is designed for parallelized and simultaneous cultivation of up to 24 different types of cells or a single type of cell at 24 different conditions. Twenty-four culture vessels of about 200 ml are arranged in four columnssix rows. The system is equipped with four independent thermostated waterbaths, each of which accommodates six culture vessels. A two-channel liquid handler is attached in order to distribute medium from the reservoir to the culture vessels, to transfer seed or other reagents, and to take an aliquot from the growing cells. Cells in each vessel are agitated and aerated by sparging filtered air. We tested the system by growing Escherichia coli BL21(DE3) cells harboring a plasmid for a model protein, and used it in optimizing protein expression conditions by varying the induction temperature and the inducer concentration. The results revealed the usefulness of our custom-made cell cultivation robot in screening optimal conditions for the expression of soluble proteins.

Strain , a pale yellow-pigmented bacterium and new isolate from deep subsurface water of the South Coast of Korea, was subjected to a polyphasic taxonomic study. grew between 5 and , pH 5.3-10.5, and tolerated up to 13% NaCl. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain was associated with the genus Arthrobacter and phylogenetically closely related to the type strains Arthrobacter tumbae (99.4%) and Arthrobacter parietis (99.1%). However, DNA-DNA hybridization experiments revealed 2.1% and 12% between strain and Arthrobacter tumbae and Arthrobacter parietis, respectively. Thus, the phenotypic and phylogenetic differences suggested that should be placed in the genus Arthrobacter as a novel species, for which the name Arthrobacter subterraneus sp. novo is proposed. In addition, the type strain for the new species is ().

This study was conducted to find the optimum extraction condition of Gold-Thread for antibacterial activity against Streptococcus mutans using The evolutionary operation-factorial design technique. Higher antibacterial activity was achieved in a higher extraction temperature () and in a longer extraction time (). Antibacterial activity was not affected by differentiation of the ethanol concentration in the extraction solvent (). The maximum antibacterial activity of clove against S. mutans determined by the EVOP-factorial technique was obtained at extraction temperature, 26 h extraction time, and 50% ethanol concentration. The population of S. mutans decreased from 6.110 logCFU/ml in the initial set to 4.125 logCFU/ml in the third set.

The tissue blot immunobinding assay (TBIA) is widely used for the detection and localization of plant viruses in various plant tissues. The basic experimental procedures of TBIA sampling and blotting were simplified using commercially available micropipette tips. This method was termed the ring-blot immunobinding assay (R-BIA), as the blot on the membrane forms a ring shape. The detection efficacy of R-BIA was tested for two chili pepper viruses, pepper mild mottle tobamovirus (PMMoV) and pepper mottle potyvirus (PepMoV), following the optimized serological procedures of TBIA (length of the incubation period and BSA concentration, and primary and secondary antibodies). Sensitivity of the R-BIA was about 1 ng/ml of purified PMMoV in pepper leaf sap from a healthy pepper plant. R-BIA also showed high specificity in the detection of PMMoV and PepMoV. Moreover, the modified sampling and blotting procedures were simpler and more reliable than other TBIA methods (such as whole-leaf blotting and crushed-leaf blotting), suggesting that the R-BIA may be used for medium- to large-scale detection of plant viruses in laboratories with minimal facilities.

A bacterium, Burkholderia sp. JBA3, which can mineralize the pesticide parathion, was isolated from an agricultural soil. The strain JBA3 hydrolyzed parathion to p-nitrophenol, which was further utilized as the carbon and energy sources. The parathion hydrolase was encoded by a gene on a plasmid that strain JBA3 harbored, and it was cloned into pUC19 as a 3.7-kbp Sau3AI fragment. The ORF2 (ophB) in the cloned fragment encoded the parathion hydrolase composed of 526 amino acids, which was expressed in E. coli DH10B. The ophB gene showed no significant sequence similarity to most of other reported parathion hydrolase genes.

Cassiae Semen (seeds of Cassia tora) showed a remarkably different HPLC chromatogram after being treated with a crude enzyme extract from Aspergillus usamii. Increased and decreased compounds were identified as aurantio-obtusin and glucoaurantio-obtusin, respectively. The aurantio-obtusin content reached its maximum level ( extract) after being incubated for 50 min at , whereas the inactivated crude enzyme-treated control remained unchanged (). On the other hand, the glucoaurantio-obtusin content decreased by less than one-third () ofthe untreated control (), suggesting that an increase in aurantio-obtusin content originated from the enzymatic cleavage of its glucoside glucoaurantio-obtusin.

We attempted to modulate the overall protein expression rate through the addition of a repressor against the araBAD promoter system of Escherichia coli, in which glucose was used as a repressor. Therefore, 0.5% L-arabinose was initially contained as an inducer in culture medium, and either 2% glucose or 2% glycerol was used as a carbon source, and it was found that the expression of recombinant interferon- could be observed at the beginning of the batch culture when glycerol was used as a carbon source. However, when glucose was used, the initiation of recombinant interferon- expression was delayed compared with that when glycerol was used. Furthermore, when the addition of 0.5% glucose was carried out once or twice after 0.5% L-arabinose induction during DO-stat fed-batch culture, the distributions of soluble and insoluble recombinant interferon- were modulated. When glucose was not added after the induction of L-arabinose, all of the expressed recombinant interferon- formed an inclusion body during the later half of culturing. However, when glucose was added after induction, the expressed recombinant interferon- did not all form an inclusion body, and about half of the total recombinant interferon- was expressed in a soluble form. It was deduced that the addition of glucose after the induction of L-arabinose might lower the cAMP level, and thus, CAP (catabolite activator protein) might not be activated. The transcription rate of recombinant interferon- in the araBAD promoter system might be delayed by the partial repression. This inhibition of the transcription rate probably resulted in more soluble interferon- expression caused by the reduction of the protein synthesis rate.

The aim of this study was to develop a new fermentation method in order to improve the digestion of soybean protein, and to promote normal fermentation of soybean. A proximate composition, such as moisture, pH, and reducing sugar, of fermented soybeans by the new fermentation was similar to those of controls. Neutral protease activity, the most important factor for fermented soybean products, was the highest, having about 636 Dig at 54 h fermentation. The content of total free amino acid was almost 3-18 times higher than controls. The three-step fermented soybeans can be used as a functional food ingredient for human consumption, with higher protein digestibility.